Escherichia coli K1 is the most common cause of meningitis in neonates. Ineffectiveness of antibiotic therapy over the last few decades and the emergence of antibiotic resistant E. coli strains imply that there is a great unmet need for new methods of treatment and prevention. Incomplete understanding of the mechanisms involved at every step of pathogenesis is attributed to this poor outcome. For example, the mechanisms by which E. coli K1 enters the human brain microvascular endothelial cells (HBMEC) that constitute the BBB and disrupts tight junctions (TJs) are poorly understood. We have established that outer membrane protein A (OmpA) of E. coli interacts with endothelial cell gp96 (Ecgp);a receptor specifically expressed on HBMEC, to invade and disrupt the TJs. The importance of OmpA-Ecgp interaction is further supported by our findings that 1) E. coli strains that either lack OmpA or express non-functional OmpA do not induce meningitis in a newborn mouse or rat model and 2) Mice in which Ecgp expression was suppressed were resistant to E. coli infection. Intriguingly, OmpA interaction with Ecgp triggers the production of nitric oxide (NO) due to iNOS activation and thereby enhances the expression of the receptor to allow the bacteria to invade more efficiently. In agreement, iNOS-/- mice are resistant to E. coli infection and also administration of an iNOS specific inhibitor, aminoguanidine (AG), during high-grade bacteremia prevented the occurrence of meningitis. Novel computer modeling methods were utilized to study the interaction of OmpA and Ecgp and to identify small molecule inhibitors that prevent the E. coli invasion of HBMEC. Three small molecules exhibited more than 80% inhibition of E. coli invasion in HBMEC both in vitro and in vivo. Our studies have also revealed that Ecgp interaction with Robo4 at the HBMEC membrane increases upon infection with E. coli. Further, a GTPase activating protein, IQGAP1, which binds both actin and b-catenin, appears to play a role in the invasion process. IQGAP1 is a client protein for Stat3, which was shown to be associated with Ecgp, indicating that IQGAP1 might be relaying Ecgp mediated signals to induce E. coli invasion. Thus, our hypothesis is that the interaction of OmpA and Ecgp is fundamental to initiate signaling events that induce E. coli invasion and increased permeability of the BBB. In Aim 1, we propose to define the binding domains of Ecgp that orchestrate the interaction of Ecgp/Robo4 with OmpA. Next, to understand whether Ecgp interaction with Robo4 contributes to signaling events to induce NO production and thereby modulating IQGAP1 association with b-CAT to dislodge it from TJs will be tested in Aim 2. Then, in Aim 3 we will modify the antagonists for higher inhibition efficiency and couple them to nanoparticles, which will carry a load of iNOS inhibitors to deliver to brain to prevent E. coli induced meningitis in newborn rats. Translational medicine is the outcome of this application in which studies of basic biology and applied technology to develop new strategies of prevention will be integrated. PUBLIC HEALTH RELEVANCE: Ineffectiveness of antibiotic therapy over the last few decades and the emergence of multidrug resistant strains make the neonatal meningitis due to E. coli K1 a serious health problem;and imply that there is a great unmet need for new methods of treatment and prevention. Our studies have demonstrated that the interaction of OmpA and endothelial cell gp96 (Ecgp) is fundamental to initiate signaling events that induce E. coli invasion and increased permeability of the blood-brain barrier (BBB). In this application, we will dissect the structural features of Ecgp that promote signaling events necessary for the invasion process and to breach the BBB permeability, which information will be used to develop Ecgp antagonists to couple to nanoparticles for targeted delivery to brain for preventing E. coli meningitis in newborn animals.